Eukaryotic cells have developed sophisticated defenses aimed at limiting viral replication and thereby preventing infection from escalating to other cells. Among the many interferon-stimulated genes whose expression is up-regulated in the antiviral response is viperin (Virus Inhibitory Protein; Endoplasmic Reticulum associated, INterferon inducible), which has been shown to restrict the infectivity of a number of important human viruses including influenza A, HIV and hepatitis C. One of the most interesting features of viperin is that it appears to be a member of the radical SAM enzyme family; these enzymes reductively cleave S- adenosylmethionine to generate an adenosyl radical that is essential for catalysis. The involvement of radical SAM chemistry in the mammalian antiviral response was completely unexpected, as the radical SAM enzymes so far studied have almost exclusively been involved in microbial metabolism. Studies in mammalian cells have implicated a number of proteins, both cellular and viral, as targets of viperin. However, none of these interactions have been characterized directly and the mechanism(s) by which viperin inhibits its target enzymes, including the role of radical SAM chemistry in the reaction, remain unknown. We propose to study the interaction of viperin with farnesyl pyrophosphate synthase (FPPS), a key enzyme in the mevalonate biosynthetic pathway and the best-characterized target of viperin. Based on our preliminary data and well-documented protein modifications catalyzed by other radical SAM enzymes, we hypothesize that viperin inactivates FPPS by covalent modification, e.g. peptide-backbone cleavage, leading to its degradation. The project's goals are to determine the mechanism by which viperin inhibits FPPS, and then to extend these studies to other enzyme targets to establish whether viperin inhibits its targets by a common mechanism. The research will take a two-pronged approach that will combine studies on purified enzymes in vitro with studies on immuno-tagged enzymes transfected in mammalian cell lines. In vivo studies aim to evaluate the regulation of FPPS activity by viperin under physiological conditions. Targeted proteomics approaches will be used to identify other potential targets of viperin and to detect potential covalent modifications of FPPS by viperin in vivo. Targeted metabolomics approaches will be used to search for the products of radical SAM chemistry in vivo and to examine the perturbation of metabolites levels in the mevalonate pathway arising from inhibition of FPPS. In vitro studies will focus on determining in detail the mechanism by which viperin harnesses radical SAM chemistry to inactivate FPPS. Informed by the results of experiments on FPPS, the studies will be extended to examine the interaction of viperin with other target enzymes, including the mitochondrial trifunctional protein, which is involved in the catabolism of fatty acids by the -oxidation pathway.